Abstract Recent epidemics of chikungunya virus (CHIKV), Ebola virus or Zika virus have shown how viruses previously considered a low threat can emerge suddenly, destroying lives and economies. Multivalent treatments or vaccines that are effective against diverse virus strains are needed to deal with emerging threats before they reach epidemic status. In this proof of concept project, we will use a protein structure- and epitope- based approach to construct a multivalent vaccine against two quite diverse mosquito borne alphaviruses, both severely effecting human health and with periodic outbreaks in the Americas, including the US, Venezuelan equine encephalitis virus (VEEV), which has a high mortality in zoonotic outbreaks in humans, and CHIKV, which can cause chronic arthralgia. We hypothesize that an optimized attenuated virus, expressing mosaic envelope proteins projecting epitopes from both CHIKV and VEEV, will induce protective antibodies against both parent viruses as well as a wide spectrum of alphaviruses. A vaccine incorporating this would substantially reduce the cost and regulatory burden compared to the current ?cocktail? approach, where individual viruses are combined. The envelope E2 proteins will be designed using structural and computational tools, developed by the PI and his collaborators on projects with measles, flaviviruses, filovirus and alphaviruses, and incorporated into vaccines developed in Dr. Weaver?s group. Together, we previously showed that a consensus E2 representing several different VEEV strains could be incorporated into infectious virus. Even without optimization, the consensus virus vaccine protected mice against a wild type (wt) strain of VEEV nearly as well as those vaccinated with the same wt strain. Our first aim is to express and characterize multivalent, physicochemical consensus proteins of the E2 glycoprotein that will represent both CHIKV and VEEV strains. These will be optimized to bind antibodies against diverse alphaviruses and generate cross protective antibodies in mice. A concurrent second aim is to introduce epitopes known to be the binding site for neutralizing antibodies into the wt-E2 proteins of CHIKV and VEEV, expressed in the context of a whole virus, and determine the range of protection these provide. In a third aim, the optimized E2 proteins will be incorporated into a novel whole virus vaccine candidate based on the alphavirus Eilat (EILV), which replicates in mosquito but not mammalian cells. Vaccinated mice will be challenged with diverse strains of CHICV, VEEV, and other alphaviruses. Dr. Weaver?s group has shown that replacing the E proteins of EILV with those of CHIKV or VEEV can lead to new diagnostic tools and an EILV/CHIKV vaccine candidate that remains restricted in its growth to insect cells but generates protection against CHIKV. The anticipated outcome will be an optimized, broadly protective vaccine against diverse alphavirus strains. The optimized, multivalent E2 proteins can also be inserted into other vaccine platforms, such as alpha-virus replicon particles or DNA-based vaccines.